Human blood and lymph contain various types of cells and each cell plays important roles. For example, the erythrocyte carries oxygen; platelets have hemostatic action; and lymphocytes prevent from infection. These various cells originate from hematopoietic stem cells in the bone marrow. Recently, it has been clarified that the hematopoietic stem cells are differentiated to various blood cells, osteoclasts and mast cells by stimulation of various cytokines in vivo and environmental factors. In the cytokines, there have been found, for example, erythropoietin (EPO) for differentiation to erythrocytes; granulocyte colony-stimulating factor (G-CSF) for differentiation to leukocytes; and platelet growth factor (mpl ligand) for differentiation to megakaryocytes which is a platelet producing cells, and the former two have already been clinically applied.
The undifferentiated blood cells are generally classified into two groups consisting of blood precursor cells which are destined to differentiate to specific blood series and hematopoietic stem cells which have differentiation ability to all series and self-replication activity. The blood precursor cells can be identified by various colony assays, however identification method for the hematopoietic stem cells has not been established. In these cells, stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-6 (IL-6), interleukin-1 (IL-1), granulocyte colony stimulating factor (G-CSF) and oncostatin M have been reported to stimulate cell differentiation and proliferation.
Trials for expansion of hematopoietic stem cells in vitro have been examined in order to replace bone marrow transplantation for applying hematopoietic stem cell transplantation therapy or gene therapy. However, when the hematopoietic stem cells are cultured in the presence of the above mentioned cytokines, multi-differentiation activities and self-replication activities, which are originally in the position of the hematopoietic stem cells, gradually disappeared and are changed to the blood cell precursors which are only to differentiate to specific series after 5 weeks of cultivation, and multi-differentiation activity which is one of the specific features of the hematopoietic stem cells, is lost (Wagner et al. Blood 86, 512-523, 1995).
For proliferation of the blood precursor cells, single cytokine is not sufficient to effect, but synergistic action of several cytokines are important. Consequently, in order to proliferate the hematopoietic stem cells in maintaining with specific features of the hematopoietic stem cells, it is necessary to add cytokines which suppress differentiation together with the cytokines which proliferate and differentiate the undifferentiated blood cells. In general, many cytokines, which stimulate proliferation or differentiation of cells, are known, but small numbers of cytokines, which suppressed cell differentiation, are known. For example, leukemia inhibitory factor (LIF) has an action of proliferation of mouse embryonic stem cells without differentiation, but it has no action against the hematopoietic stem cells or blood precursor cells. Transforming growth factor (TGF—β) has suppressive action for proliferation against various cells, but no fixed actions against the hematopoietic stem cells or blood precursor cells.
Not only blood cells but also undifferentiated cells, especially stem cells are thought to be involved in tissue regeneration. These regeneration of tissues and proliferation of undifferentiated cells in each tissue can be applied in various ways by referring to the known reference (Katsutoshi Yoshizato, Regeneration—a mechanism of regeneration, 1996, Yodosha Publ. Co.).
Notch is a receptor type membrane protein, which involves in regulation of nerve cells differentiation found in Drosophila. Homologues of the Notch are found in various animal kinds exceeding to the invertebrate and vertebrate including nematode (Lin-12). Xenopus laevis (Xotch), mouse (Motch) or human (TAN-1).
Ligand of the Notch in Drosophila is known. These are Drosophila Delta (Delta) and Drosophila Serrate (Serrate). Notch ligand homologues are found in various animal kinds as similar to the Notch of receptors (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).
Human Notch homologue, TAN-1 is found widely in the tissues in vivo (Ellisen et al., Cell 66, 649-661, 1991). Three Notch analogous molecules other than TAN-1 are reported (Artavanis-Tsakonas et al., Science 268, 225-232, 1995). Expression of TAN-1 was also observed in CD34 positive cells in blood cells by PCR (Polymerase Chain Reaction) (Milner et al., Blood 83, 2057-2062, 1994). However, in relation to humans, gene and amino acid sequences of human Delta and human Serrate, which are thought to be the Notch ligand, have not been reported as scientific reports in April 1997.
In Drosophila Notch, binding with the ligand was studied and investigated in details, and it was found that the Notch can be bound to the ligand with Ca++ at the binding region, which is a repeated amino acid sequence No. 11 and No. 12 in the amino acid sequence repeat of Epidermal Growth Factor (EGF) like repeating (Fehon et al., Cell 61, 523-534, 1990, Rebay et al., ibid. 67, 687-699, 1991 and International Publication WO 92/19734). EGF-like repeated sequences are conserved in Notch homologues of the other species. Consequently, the same mechanism in binding with ligand is estimated. An amino acid sequence which is called as DSL (Delta-Serrate-Lag-2) near the amino acid terminal, and EGF-like repeated sequence as like in the receptor are conserved in the ligand (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).
EGF-like sequence has been found in thrombomodulin (Jackman et al., Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986), low density lipoprotein (LDL) receptor (Russell et al., Cell 37, 577-585, 1984), and blood coagulating factor (Furie et al., Cell 53, 505-518, 1988), and is thought to play important roles in extracellular coagulation and adhesion.
Recently, the vertebrate homologues of the cloned Drosophila Delta were found in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1), and it has reported that X-Delta-1 had acted through Xotch in the generation of the protoneuron (Henrique et al., Nature 375, 787-790, 1995 and Chitnis et al., ibid. 375, 761-766,1995). Vertebrate homologue of Drosophila Serrate was found in rat as rat Jagged (Jagged) (Lindsell et al., Cell 80, 909-917, 1995). According to the Lindsell et al., mRNA of the rat Jagged is detected in the spinal cord of fetal rats. As a result of cocultivation of a myoblast cell line that is forced excess expressed rat Notch with a rat Jagged expression cell line, suppression of differentiation of the myoblast cell line is found. However, the rat Jagged has no action against the myoblast cell line without forced expression of the rat Notch.
A hypothesis has been set up so that Notch and its ligand have an action of differential regulation not only for neuroblasts and myoblasts, but also for various undifferentiated cells, especially blood undifferentiated cells. However, as far as clinical applications in humans, prior known different species such as chicken or Xenopus laevos type Notch ligand have problems with species specificities and antigenicities. Consequently, obtaining prior unknown human Notch ligand is essentially required. The inventor suspected that a molecule having DSL domain and EGF-like domain which are common to Notch ligand molecules and a ligand of the human Notch (TAN-1 etc.), which is a human Delta homologue (hereinafter designates as human Delta) and human Serrate homologue (hereinafter designates as human Serrate), may be found. In addition, these findings may be a candidate for a drug useful for differential regulation of undifferentiated cells.
As a result, in the previous patent application, a gene cloning of three types of molecules including human Delta-1, human Serrate-1 and human Serrate-2 molecules as the human Notch ligand molecules was made, and it was found that these molecules have an action on blood undifferentiated cells. (Refer to WO 97/19172 Differentiation-suppressive polypeptide and WO 98/02458 Differentiation-inhibitor). As for the human Notch ligand molecule, according to the recent report, partial gene and partial amino acid sequences of the human Delta-1 like molecule, which are, however, incomplete with respect to the specification and disclosure of the full-length sequence, have been disclosed in the International Publication WO 97/01571. Further, WO 96/27610 discloses total length gene and total length amino acid sequences of human Serrate-1 (humanJagged-1). Also, WO 96/27610 discloses partial length gene and partial length amino acid sequences of human Serate-2 (human Jagged-2). This gene sequence might have erroneous sequences and this gene sequence generates frame shift, which results completely different amino acid sequence of our WO 98/02458, Differentiation-inhibitor. In addition, the said prior arts did not disclose gene cloning of amino terminals. Consequently, the gene sequences and amino acid sequences are incomplete. As a result of searching the gene sequence database, Genebank Release 98 (December 1996), there are four entries about human Serrate-1, i.e. Registered No. HSU61276, HSU3936, HSU77720 and HSU77914, however no other human Notch ligand molecules are found in the said database.
The present invention elucidates the gene sequence and amino acid sequence of novel Notch ligand molecules. Novel Notch ligand molecules and novel therapeutic uses for these molecules are also provided.
In order to search novel human Notch ligands, cross hybridization using the human Delta-1 gene was performed.
To obtain the human Delta-1 gene, methods used in the referential examples 1 and 2, and WO 97/19172 can be applied. Transformed cells, in which a vector pUCDL-1 containing cDNA coding total amino acid sequence of human Delta-1, i.e. DNA containing sequence from No. 179 to No. 2347 in SEQ ID NO: 8, is inserted into E. coli JM109, have been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, in Higashi 1-1-3, Tsukuba, Ibaragi-ken, Japan as permanent culture collection E. coli: JM109-pUCDL-1F. Date of deposition was Oct. 28, 1996 as deposition No. FERM BP-5728.
Various lengths of partial genes of this human Delta-1 gene were prepared. Using these partial lengths of genes as probes, numerous cDNA libraries were screened under various hybridization conditions to determine novel Notch ligands by cross hybridization methods.
As a result of extensive studies, the isolation of cDNA coding amino acid sequences of novel human Delta-2 have been achieved, a novel molecule having DSL domain common to Notch ligand molecules from human fetal lung cDNA library, and have prepared the expression systems of protein having various forms using the cDNA. Also we have established purification methods of the proteins which were purified and isolated.
Amino acid sequences of novel human Delta-2 are shown in the sequence listings, SEQ ID NO: 1-3. DNA sequence coding these sequences is shown in the sequence listing, SEQ ID NO: 4.
Physiological actions of the these prepared proteins were searched by using many types of cells, for example nerve undifferentiated cells, preadipocytes, hepatocytes, myoblasts, skin undifferentiated cells, blood undifferentiated cells and immune undifferentiated cells. As a result, it has been found that novel human Delta-2 had a differentiation-suppressive action against undifferentiated blood cells, and had a physiological action to maintain an undifferentiated state. Further, it has been found that the molecule has growth suppressive action against vascular endothelial cells.
No significant toxic actions were noted in the toxicity studies on mice, and useful pharmaceutical effects were suggested. Consequently, the pharmaceutical preparations containing the molecule of the present invention, medium containing the molecule of the present invention, and the device immobilized with the molecule of the present invention are novel drugs and medical materials which can maintain the blood undifferentiated cells in the undifferentiated condition. Antibody against human Delta-2 is prepared by using antigen of the said human Delta-2, and purification method of the said antibodies is established. The present invention has completed accordingly.
The present invention relates to a polypeptide comprising at least amino acid sequence of SEQ ID NO: 1 of the sequence listing, a polypeptide comprising at least amino acid sequence of SEQ ID NO: 2 of the sequence listing, and a polypeptide comprising at least amino acid sequence of SEQ ID NO: 3 of the sequence listing. The present invention also relates to the said polypeptides having differentiation suppressive action against undifferentiated cells, the said polypeptides in which the undifferentiated cells are undifferentiated cells except for those of brain and nervous system or muscular system, the said polypeptides in which the undifferentiated cells are undifferentiated blood cells, and the said polypeptides acting on vascular cells. The present invention also relates to a pharmaceutical composition comprising the said polypeptides, and the said pharmaceutical composition having differentiation suppressive action against cells, the said pharmaceutical composition in which the cells are undifferentiated blood cells, and the said pharmaceutical composition having regulatory action against vascular cells. The present invention further relates to a cell culture medium comprising the said polypeptides, the cell culture medium in which the cell is undifferentiated blood cell, and a material having immobilized thereto the polypeptide. Further, the present invention relates to a method for culturing cells using the cell culture medium or the material, and the method in which the cells are undifferentiated blood cells.
The present invention further relates to a DNA coding at least an amino acid sequence of the sequence listing, SEQ ID NO: 1, said DNA coding at least an amino acid sequence of the sequence listing, SEQ ID NO: 2, the DNA coding at least an amino acid sequence of the sequence listing, SEQ ID NO: 3, the DNA having a base sequence from 355 to 927 of the sequence listing, SEQ ID NO: 4, the DNA having base sequence from 355 to 1854 of the sequence listing, SEQ ID NO: 4 and the DNA having base sequence from 355 to 2331 of the sequence listing SEQ ID NO: 4. The present invention still further relates to a recombinant DNA comprising a DNA selected from the group consisting of the DNAs having ligated to a vector DNA which can express said DNA in the host cell, a cell transformed by the recombinant DNA, a method for culturing human cells with the said cells, and a process for production of said polypeptide by culturing the said cells and isolating the compound produced in the cultured mass. The present invention still more further relates to an antibody specifically recognizing a polypeptide having an amino acid sequence of the sequence listing, SEQ ID NO: 3.